Human immunosuppressive protein

ABSTRACT

A composition of an immunosuppressant protein (HISP) which is achieved by the steps of obtaining supernatant from hNT neuronal cells; exposing the supernatant to preparative polyacrylamide gel; placing the active isoelectric fraction on a Blue Sepharose column to bind albumin; and collecting the free fraction containing the concentrated, isolated HISP. The HISP is anionic, has a molecular weight of 40-100 kDa, an isoelectric point of about 4.8 and is obtained from the supernatant of hNT cells. HISP can suppress proliferation of responder peripheral blood mononuclear cells in allogeneic mixed lymphocyte cultures; HISP can suppress T-cell proliferation and IL-2 production in response to phorbol 12-myristate 13-acetate (PMA), ionomycin and concanavalin-A. HISP does not act through the T-cell receptor-CD3 complex or via altered accessory signal cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of pending U.S. Ser. No.10/621,061, filed Jul. 16, 2003, which claims the benefit of U.S.Provisional Application No. 60/396,928, filed Jul. 16, 2002.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Some of the work disclosed herein was supported by Small BusinessTechnology Research Grant No. 1-R41-AI50367-01 from the NationalInstitute of Allergy and Infectious Diseases. The government has certainrights in this invention.

BACKGROUND

1. Technical Field

The invention is in the fields of drug therapy; more specifically, theinvention encompasses a protein for immunosuppression.

2. The Prior Art

The central nervous system (CNS) actively maintains immune privilege(Carson and Sutcliffe, 1999; Fabry et al., 1994), in part by restrictingimmune cell access (Goldstein and Betz, 1986; Hickey et al., 1991),having limited afferent antigen drainage (Weller et al., 1996; Cserr andKnopf, 1992), locally suppressing immune responsiveness (Irani et al.,1996; Irani et al., 1997), guiding the recruitment and differentiationof effector cell phenotypes (Aloisi et al., 1998; Carson et al., 1999),and possessing weak antigen presenting cells (Carson et al., 1998).

Neurons may directly modulate immune responsiveness. Absence ofconstitutive neuronal MHC expression may limit anti-neuronal cytotoxicT-cell effector mechanisms (Rall, 1998). Glycosphingolipids known asgangliosides are enriched within neurons, can be shed from the cellsurface, are immunosuppressive, and may contribute to immune privilege(Irani et al., 1996; Rall, 1998). Gangliosides suppress the expressionof MHC molecules (Massa, 1993), the proliferation of T-cells, and theproduction of IL-2 (Irani et al., 1996; Irani et al., 1997; Bergelson,1995; Robb, 1986; Dyatlovitskaya and Bergelson, 1987).

The therapeutic approach of transfecting and transplanting neurons toameliorate neurological deficits requires a defined, preferably clonalsource of differentiated human neurons amenable to efficienttransfection and sustained expression of therapeutic genes (Trojanowskiet al., 1997; Cook et al., 1994). A therapeutic effect is anticipatedshould the engrafted cells retain a neuronal phenotype, functionallyintegrate, and deliver a sustained level of therapeutically relevantprotein to the affected region of the brain (Cook et al., 1994). Thisapproach has evolved from trials utilizing neuronal isolates of theembryonic ventral mesencephalon (Kordower et al., 1995; Bjorklund, 1992;Perlow et al., 1979), modified neuronal progenitors (Sabate et al.,1995), neurons (Anton et al., 1994), or fibroblasts (Fisher et al.,1991). Ganglioside shedding and the absence of MHC expression may favorresistance of the neuronal graft to MHC-restricted T-cell attack(Lampson and Siegel, 1988).

Embryonic neurons as grafts are limited by their heterogeneity, expense,scarcity, diminishing viability over time, and refractoriness tostandard transfection techniques (Cook et al., 1994; Meichsner et al.,1993). A promising alternative neuron, which is amenable totransfection, is derived from the embryonal carcinoma cell lineNtera2/D1, a putative neuronal progenitor (Cook et al., 1994; Andrews etal., 1984). Ntera2/D1 differentiate in response to treatment withall-trans-retinoic acid into a mixture of cells, including postmitoticcells with a neuronal phenotype (Andrews, 1984; Pleasure et al., 1992;Pleasure and Lee, 1993). Cultures are selectively enriched forNtera2/D1-derived neurons (designated hNT neurons) by inhibiting thenon-neuronal cells with mitotic inhibitors, and by replating hNT neuronson poly-D-lysine plus laminin, which encourages growth of polarizedprocesses. In this manner, cultures comprised of >90% hNT neurons areprepared (Cook et al., 1994).

hNT neurons have identifiable axons and dendrites (Andrews, 1984),retain a plasticity to regenerate and extend neurites after multiplereplatings in vitro (Cook et al., 1994), and express neurofilamentscharacteristic of neuronal development and the adult CNS (Andrews, 1984;Lee and Andrews, 1986). hNT neurons synthesize neurotransmitters,express the catecholamine biosynthetic enzyme tyrosine hydroxylase, andexcrete the dopamine metabolite homovanillic acid (Zeller and Strauss,1995; lacovitti and Stull, 1997). Transplanted hNT neurons are capableof long-term functional integration (Kleppner et al., 1995), arenontumorigenic (Trojanowski et al., 1997), and can correct behavioraldeficits in the lesioned rodent (Borlongan et al., 1998).

Although a therapeutic potential of hNT neuronal grafts has beenimplied, a paucity of data exists regarding its MHC and immunologicalfeatures. Retinoic acid-induced differentiation of Ntera2/D1 causes theproduced hNT neurons to express MHC class I and β-2 microglobulinmolecules (Segars et al., 1993), but whether hNT neurons express adiscernable MHC phenotype that can activate allogeneic immunocytes hasnot been determined. An increase in the expression of gangliosides(e.g., GD₃ and GT₃) and the glycolipid sialyltransferases thatcontribute to their synthesis occurs during the differentiation of someembryonal carcinoma cells (Chen et al., 1989; Osania et al., 1997).Whether hNT neurons can modulate immune responses and shed gangliosidesat immunosuppressive levels have not been determined. Some CNS neoplasms(e.g., gliomas) express immunosuppressive levels of transforming growthfactor-β. (TGF-β) (Weller and Fontana, 1995). TGF-β inhibits T-cellproliferation by suppressing IL-2-mediated proliferative signals (Ahujaet al., 1993). Retinoic acid treatment increases TGF-β expression duringmurine embryogenesis (Mahmood et al., 1995), and during embryonalcarcinoma cell differentiation (Rizzino et al., 1983), but whether hNTneurons express immunosuppressive levels of TGF-β has not beendetermined.

SUMMARY OF THE INVENTION

In one embodiment, there is disclosed a method for purifying animmunosuppressant protein (HISP) which has the following steps: a)obtaining supernatant from hNT cells; b) exposing the supernatant topreparative polyacrylamide gel electrophoresis to produce 20 isoelectricfractions, including active isoelectric fraction #10; c) placing theactive isoelectric fraction on a Blue Sepharose column to bind albumin;and d) collecting the free fraction containing the concentrated,isolated HISP.

Also disclosed is the compound produced by claim 1.

In another embodiment, there is a method of treating inflammation, themethod comprising administering an effective amount of animmunosuppressant protein (HISP).

In yet another embodiment, there is an isolated immunosuppressantprotein (HISP), the protein comprising an anionic protein and having amolecular weight of 40-100 kDa; having an isoelectric point of about4.8; being obtained from hNT cell supernatant; not being obtained fromNCCIT embryonal carcinoma cells, T98G glioblastoma cells or THP-1monocytic leukemia cells; losing activity when treated with heat, pH 2,pH 11, or mixed with trypsin or carboxypeptidase; losing no activitywhen incubated with neuraminidase; being capable of suppressingproliferation of responder peripheral blood mononuclear cells inallogeneic mixed lymphocyte cultures; being capable of suppressingT-cell proliferation and IL-2 production in response to phorbol12-myristate 13-acetate (PMA), ionomycin and concanavalin-A; beingcapable of maintaining T cells in a quiescent G₀/G₁ state withoutlowering their viability; not binding to heparin-sepharose CL-B gel; notbinding to albumin-binding resin Blue Sepharose; concentrating with YM10ultrafiltration; and not acting through the T-cell receptor-CD3 complexor via altered accessory signal cells.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIG. 1 shows the result of ethidium bromide-stained gel electrophoresisof the amplified products of MHC class I specific polymerase chainreactions using Ntera2/D1 genomic DNA as template revealed products of630 (A1), 350 (Bw6), 605 and 415 (B8), and 1060 (Cw7) base pairs,indicating a MHC class I genotype of A1 B8 Bw6 Cw7.

FIGS. 2A and 2B are graphs showing the quantity of ³H-TdR incorporationby control and PHA-activated cells. FIG. 2A shows that the hNT neuronsdid not activate allogeneic PBMC to proliferate in mixedlymphocyte-neuron cultures (MLNC) compared to the proliferation ofallogeneic mixed lymphcyte cultures (MLC). When hNT supernatant wasadded the proliferation of allogeneic MLC was suppressed to basal levelscomparable to those of unstimulated controls. Values are mean±SD. FIG.2B shows that the hNT supernatant significantly suppressed theproliferation of PHA-stimulated PBMC. Supplemental IL-2 augmented thePHA-induced proliferation of PBMC, but did not rescue the T-lymphocytessuppressed by exposure to hNT supernatant. Values are mean±SD.

FIG. 3 shows that the mean±SD levels of IL-2 expressed by PHA-stimulatedPBMC were significantly less when cultured in the presence of hNTsupernatant (▪) compared to controls (⋄), 48 hours after mitogenstimulation (p<0.01).

FIG. 4 shows the results when hNT supernatant was concentrated and theconcentrate fractionated. The fractions from gel filtration wereevaluated for protein content (continuous tracing), and assessed for anability to suppress the PHA-stimulated proliferation of T-cells(vertical bars). Suppression of T-cell proliferation was greatest infractions corresponding to a mass of 40-100 kDa.

FIG. 5 shows the peak active fraction of hNT supernatant from gelfiltration which underwent isoelectric focusing using a narrow ampholyterange of pH 4-6 (⋄). Isoelectric tractions were assessed for proteincontent at 280 nm (●). Of the twenty fractions collected and evaluated,only isoelectric fraction #10 (IEF-10) significantly suppressed theproliferation of PBMC induced by PHA (vertical slashed bar) (p<0.01).Values are mean±SD.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

In light of the therapeutic potential of hNT neuronal grafts, weevaluated hNT for its MHC and immunological characteristics, and forevidence of neuronal regulation of immune cells in vitro. During thisevaluation, we quite serendipitously discovered a novel hNTneuron-expressed immunosuppressive protein (HISP) with characteristicsunlike gangliosides or TGF-.beta., which is potently suppressive ofT-cell activation, proliferation, and the production of IL-2.Consequently, hNT neuronal grafts may prove to be both therapeutic andself-protective, engrafted alone, or as co-grafts with other neurons.

Ntera2/D1 cells had an A1 B8 Bw6 Cw7 DR3 DR52 major histocompatibilitycomplex (MHC) genotype. Its neuronal derivative, hNT neurons, expressedA1 B8 Bw6 MHC class I molecules, but did not activate, and its hNTsupernatant suppressed allogeneic mixed lymphocyte cultures (MLC)>98%(p<0.01), phytohemagglutinin (PHA)-activated T-cell proliferation >87%(p<0.01), even 48 hours after stimulation, suppressed phorbol12-myristate 13-acetate (PMA)/ionomycin-induced T-cellproliferation >99% (p<0.001), and reduced interleukin-2 (IL-2)production (p<0.01), while maintaining T-cells in a quiescent G₀/G₁state without lowering their viability. This immunosuppressive activitywas attributed to a 40-100 kDa anionic hNT protein with an isoelectricpoint of 4.8.

Immunosuppressant as used herein is a substance that prevents orattenuates immunologic phenomena. For example, such immunologicphenomena include inflammation, autoimmunity, GVHD and graft rejection.Examples of currently available immunosuppressants include but are notlimited to cyclosporine A, cyclophosphamide, prednisone and tacrolimus(FK506).

“Beneficial effect” is an observable improvement over the baselineclinically observable signs and symptoms. For example, a beneficialeffect could include improvements in graft survival, decreasedinflammation or improvements in the disease treated.

“Mammal” includes humans and other mammals that would reasonably benefitfrom treatment of immune and inflammation disorders, including pets likedogs, cats and horses.

An “active fragment” of the immunosuppressant protein is a peptide orpolypeptide that comprises a fragment of the protein and retains atleast one physiological activity of the immunosuppressant protein, e.g.,by acting as a suppressor of the T lymphocyte activation reaction.

A peptide of the present invention includes, but is not limited to,those containing, as a primary amino acid sequence, all or part of theamino acid sequence of the immunosuppressant protein including alteredsequences in which functionally equivalent amino acid residues aresubstituted for residues within the sequence resulting in a conservativeamino acid substitution. Such alterations define the term “aconservative substitution” as used herein. For example, one or moreamino acid residues within the sequence can be substituted by anotheramino acid of a similar polarity, which acts as a functional equivalent,resulting in a silent alteration. Substitutes for an amino acid withinthe sequence may be selected from other members of the class to whichthe amino acid belongs. For example, the nonpolar (hydrophobic) aminoacids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan and methionine. Amino acids containingaromatic ring structures are phenylalanine, tryptophan, and tyrosine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine. The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Such alterations will not be expected to affect apparentmolecular weight, as determined by polyacrylamide gel electrophoresis,or isoelectric point.

Particularly preferred conservative substitutions are as follows: Lysfor Arg and vice versa such that a positive charge may be maintained;Glu for Asp and vice versa such that a negative charge may bemaintained; Ser for Thr such that a free —OH can be maintained; and Glnfor Asn such that a free NH₂ can be maintained.

A polypeptide is a polymer of amino acid residues. As used herein,unless otherwise specifically indicated, the terms “polypeptide” and“protein” are used interchangeably with each other and with the term“peptide” though, the term “peptide” is preferably used for smalleramino acid polymers, e.g., less than 50 amino acids and/or for fragmentsof a protein that are missing at least about one third of their aminoacids.

One skilled in the art can readily adapt the nucleic acid sequences ofthe invention to any system that is capable of producing nucleic acidsto produce the nucleic acids of the invention. The nucleic acids of theinvention, which may optionally comprise a detectable label, may beprepared as cDNA clones, genomic clones, RNA transcribed from eithercDNA or genomic clones, synthetic oligonucleotides, and/or syntheticamplification products resulting, e.g., from PCR. The nucleic acids ofthe invention may be prepared in either single- or double-stranded form.

Synthetic peptides prepared using the well known techniques of solidphase, liquid phase, or peptide condensation techniques, or anycombination thereof, can include natural and unnatural amino acids.Amino acids used for peptide synthesis may be standard Boc (N_(α)-aminoprotected N_(α)-t-butyloxycarbonyl) amino acid resin with the standarddeprotecting, neutralization, coupling and wash protocols of theoriginal solid phase procedure of Merrifield [J. Am. Chem. Soc,85:2149-2154 (1963)], or the base-labile N_(α)-amino protected9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpinoand Han [J. Org. Chem., 37:3403-3409 (1972)]. Both Fmoc and BocN_(α)-amino protected amino acids can be obtained from Fluka, Bachem,Advanced Chemtech, Sigma, Cambridge Research Biochemical, Bachem, orPeninsula Labs or other chemical companies familiar to those whopractice this art. In addition, the method of the invention can be usedwith other N_(α)-protecting groups that are familiar to those skilled inthis art. Solid phase peptide synthesis may be accomplished bytechniques familiar to those in the art and provided, for example, inStewart and Young, 1984, Solid Phase Synthesis, Second Edition, PierceChemical Co., Rockford, III; Fields and Noble, 1990, Int. J. Pept.Protein Res. 35:161-214, or using automated synthesizers, such as soldby ABS. Thus, the ARF-p19 peptides of the invention may comprise D-aminoacids, a combination of D- and L-amino acids, and various “designer”amino acids (e.g., 13-methyl amino acids, C_(u)-methyl amino acids, andN_(α)-methyl amino acids, etc.) to convey special properties. Syntheticamino acids include omithine for lysine, fluorophenylalanine forphenylalanine, and norleucine for leucine or isoleucine. Additionally,by assigning specific amino acids at specific coupling steps, α-helices,β-turns, β-sheets, γ-turns, and cyclic peptides can be generated.

In another embodiment, the therapeutic compound can be delivered in avesicle, in particular a liposome [see Langer, Science, 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss: New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.]. To reduce its systemic side effects, this may be a preferredmethod for introducing the immunosuppressant protein.

In yet another embodiment, the therapeutic compound can be delivered ina controlled release system. For example, the polypeptide may beadministered using intravenous infusion, an implantable osmotic pump, atransdermal patch, liposomes, or other modes of administration. In oneembodiment, a pump may be used [see Langer, supra; Sefton, CRC Crit.Ref. Biomed. Eng., 14:201 (1987); Buchwald et al., Surgery, 88:507(1980); Saudek et al., N. Engl. J. Med., 321:574 (1989)]. In anotherembodiment, polymeric materials can be used [see Medical Applications ofControlled Release, Langer and Wise (eds.), CRC Press: Boca Raton, Fla.(1974); Controlled Drug Bioavailability, Drug Product Design andPerformance, Smolen and Ball (eds.), Wiley: New York (1984); Ranger andPeppas, J. Macromol. Sci. Rev. Macromol. Chem., 23:61 (1983): see alsoLevy et al., Science, 228:190 (1985); During et al., Ann. Neurol.,25:351 (1989); Howard et al., J. Neurosurg., 71:105 (1989)]. In yetanother embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., a brain tumor, thus requiringonly a fraction of the systemic dose [see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)].Preferably, a controlled release device is introduced into a subject inproximity of the site of a tumor. Other controlled release systems arediscussed in the review by Langer [Science, 249:1527-1533 (1990)].

Pharmaceutical Compositions. In yet another aspect of the presentinvention, provided are pharmaceutical compositions of the above. Suchpharmaceutical compositions may be for administration for injection, orfor oral, pulmonary, nasal or other forms of administration. In general,comprehended by the invention are pharmaceutical compositions comprisingeffective amounts of a low molecular weight component or components, orderivative products, of the invention together with pharmaceuticallyacceptable diluents, preservatives, solubilizers, emulsifiers, adjuvantsand/or carriers. Such compositions include diluents of various buffercontent (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength;additives such as detergents and solubilizing agents (e.g., Tween 80,Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), preservatives (e.g., thimerosal, benzyl alcohol) andbulking substances (e.g., lactose, mannitol); incorporation of thematerial into particulate preparations of polymeric compounds such aspolylactic acid, polyglycolic acid, etc. or into liposomes. Hylauronicacid may also be used. Such compositions may influence the physicalstate, stability, rate of in vivo release, and rate of in vivo clearanceof the present proteins and derivatives. See, e.g., Remington'sPharmaceutical Sciences, 18th Ed. [1990, Mack Publishing Co., Easton,Pa. 18042] pages 1435-1712 which are herein incorporated by reference.The compositions may be prepared in liquid form, or may be in driedpowder, such as lyophilized form.

Oral Delivery is contemplated for use herein. Oral solid dosage formsare described generally in Remington's Pharmaceutical Sciences, 18th Ed.1990 (Mack Publishing Co. Easton, Pa. 18042) at Chapter 89, which isherein incorporated by reference. Solid dosage forms include tablets,capsules, pills, troches or lozenges, cachets or pellets. Also,liposomal or proteinoid encapsulation may be used to formulate thepresent compositions (as, for example, proteinoid microspheres reportedin U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and theliposomes may be derivatized with various polymers (e.g., U.S. Pat. No.5,013,556). A description of possible solid dosage forms for thetherapeutic is given by Marshall, K. In: Modern Pharmaceutics Edited byG. S. Banker and C. T. Rhodes Chapter 10, 1979, herein incorporated byreference. In general, the formulation includes the immunosuppressantprotein (or chemically modified forms thereof) and inert ingredientswhich allow for protection against the stomach environment, and releaseof the biologically active material in the intestine.

Also specifically contemplated are oral dosage forms of the abovederivatized component or components. The component or components may bechemically modified so that oral delivery of the derivative isefficacious. Generally, the chemical modification contemplated is theattachment of at least one moiety to the component molecule itself,where said moiety permits (a) inhibition of proteolysis; and (b) uptakeinto the bloodstream from the stomach or intestine. Also desired is theincrease in overall stability of the component or components andincrease in circulation time in the body. An example of such a moiety ispolyethylene glycol or PEG.

For the component (or derivative) the location of release may be thestomach, the small intestine (the duodenum, the jejunum, or the ileum),or the large intestine. One skilled in the art has availableformulations that will not dissolve in the stomach, yet will release thematerial in the duodenum or elsewhere in the intestine. Preferably, therelease will avoid the deleterious effects of the stomach environment,either by protection of the protein (or derivative) or by release of thebiologically active material beyond the stomach environment, such as inthe intestine.

The therapeutic can be included in the formulation as finemulti-particulates in the form of granules or pellets of particle sizeabout 1 mm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs or even as tablets.The therapeutic could be prepared by compression.

One may dilute or increase the volume of the therapeutic with an inertmaterial. These diluents could include carbohydrates, especiallymannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans and starch. Certain inorganic salts may be also be used asfillers including calcium triphosphate, magnesium carbonate and sodiumchloride. Some commercially available diluents are Fast-Flo, Emdex,STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrates include but are notlimited to starch, including the commercial disintegrant based onstarch, Explotab. Binders also may be used to hold the therapeutic agenttogether to form a hard tablet and include materials from naturalproducts such as acacia, tragacanth, starch and gelatin.

An anti-frictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall. Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression also might beadded. The glidants may include starch, talc, pyrogenic silica andhydrated silicoaluminate.

In addition, to aid dissolution of the therapeutic into the aqueousenvironment a surfactant might be added as a wetting agent. Additiveswhich potentially enhance uptake of the protein (or derivative) are forinstance the fatty acids oleic acid, linoleic acid and linolenic acid.

Nasal delivery of the immunosuppressant protein or derivative thereof isalso contemplated. Nasal delivery allows the passage of the protein tothe blood stream directly after administering the therapeutic product tothe nose, without the necessity for deposition of the product in thelung. Formulations for nasal delivery include those with dextran orcyclodextran.

For nasal administration, a useful device is a small, hard bottle towhich a metered dose sprayer is attached. In one embodiment, the metereddose is delivered by drawing the pharmaceutical composition of thepresent invention solution into a chamber of defined volume, whichchamber has an aperture dimensioned to aerosolize and aerosolformulation by forming a spray when a liquid in the chamber iscompressed. The chamber is compressed to administer the pharmaceuticalcomposition of the present invention. In a specific embodiment, thechamber is a piston arrangement. Such devices are commerciallyavailable.

Alternatively, a plastic squeeze bottle with an aperture or openingdimensioned to aerosolize an aerosol formulation by forming a spray whensqueezed. The opening is usually found in the top of the bottle, and thetop is generally tapered to partially fit in the nasal passages forefficient administration of the aerosol formulation. Preferably, thenasal inhaler will provide a metered amount of the aerosol formulation,for administration of a measured dose of the drug.

Transdermal administration. Various and numerous methods are known inthe art for transdermal administration of a drug, e.g., via atransdermal patch. Transdermal patches are described in for example,U.S. Pat. No. 5,407,713, issued Apr. 18, 1995, to Rolando et al.; U.S.Pat. No. 5,352,456, issued Oct. 4, 1994, to Fallon et al.; U.S. Pat. No.5,332,213 issued Aug. 9, 1994, to D'Angelo et al.; U.S. Pat. No.5,336,168, issued Aug. 9, 1994, to Sibalis; U.S. Pat. No. 5,290,561,issued Mar. 1, 1994, to Farhadieh et al.; U.S. Pat. No. 5,254,346,issued Oct. 19, 1993, to Tucker et al.; U.S. Pat. No. 5,164,189, issuedNov. 17, 1992, to Berger et al.; U.S. Pat. No. 5,163,899, issued Nov.17, 1992, to Sibalis; U.S. Pat. Nos. 5,088,977 and 5,087,240, bothissued Feb. 18, 1992, to Sibalis; U.S. Pat. No. 5,008,110, issued Apr.16, 1991, to Benecke et al.; and U.S. Pat. No. 4,921,475, issued May 1,1990, to Sibalis, the disclosure of each of which is incorporated hereinby reference in its entirety.

It can be readily appreciated that a transdermal route of administrationmay be enhanced by use of a dermal penetration enhancer, e.g., such asenhancers described in U.S. Pat. No. 5,164,189 (supra), U.S. Pat. No.5,008,110 (supra), and U.S. Pat. No. 4,879,119, issued Nov. 7, 1989, toAruga et al., the disclosure of each of which is incorporated herein byreference in its entirety.

Pulmonary Delivery. Also contemplated herein is pulmonary delivery ofthe pharmaceutical compositions of the present invention. Apharmaceutical composition of the present invention is delivered to thelungs of a mammal while inhaling and traverses across the lungepithelial lining to the blood stream. Other reports of this includeAdjei et al. [Pharmaceutical Research, 7:565-569 (1990); Adjei et al.,International Journal of Pharmaceutics, 63:135-144 (1990) (leuprolideacetate); Braquet et al., Journal of Cardiovascular Pharmacology,13(suppl. 5): 143-146 (1989) (endothelin-1); Hubbard et al., Annals ofInternal Medicine, Vol. III, pp. 206-212 (1989) (.alpha. 1-antitrypsin);Smith et al., J. Clin. Invest., 84:1145-1146 (1989)(.alpha.-1-proteinase); Oswein et al., “Aerosolization of Proteins”,Proceedings of Symposium on Respiratory Drug Delivery II, Keystone,Colo., March, (1990) (recombinant human growth hormone); Debs et al., J.Immunol., 140:3482-3488 (1988) (interferon γ and tumor necrosis factorα); Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colonystimulating factor)]. A method and composition for pulmonary delivery ofdrugs for systemic effect is described in U.S. Pat. No. 5,451,569,issued Sep. 19, 1995, to Wong et al.

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art. With regard to construction of the delivery device,any form of aerosolization known in the art, including but not limitedto spray bottles, nebulization, atomization or pump aerosolization of aliquid formulation, and aerosolization of a dry powder formulation, canbe used in the practice of the invention.

All such devices require the use of formulations suitable for thedispensing of pharmaceutical composition of the present invention (orderivative). Typically, each formulation is specific to the type ofdevice employed and may involve the use of an appropriate propellantmaterial, in addition to the usual diluents, adjuvants and/or carriersuseful in therapy. Also, the use of liposomes, microcapsules ormicrospheres, inclusion complexes, or other types of carriers iscontemplated. Chemically modified pharmaceutical composition of thepresent invention may also be prepared in different formulationsdepending on the type of chemical modification or the type of deviceemployed.

Formulations suitable for use with a nebulizer, either jet orultrasonic, may typically comprise pharmaceutical composition of thepresent invention (or derivative) dissolved in water at a concentrationof e.g., about 0.1 to 25 mg of biologically active ingredients of apharmaceutical composition of the present invention per mL of solution.The formulation may also include a buffer and a simple sugar (e.g., forstabilization and regulation of osmotic pressure of a pharmaceuticalcomposition of the present invention). The nebulizer formulation mayalso contain a surfactant, to reduce or prevent surface inducedaggregation of the pharmaceutical composition of the present inventioncaused by atomization of the solution in forming the aerosol.

The liquid aerosol formulations contain a pharmaceutical composition ofthe present invention and a dispersing agent in a physiologicallyacceptable diluent. The dry powder aerosol formulations of the presentinvention consist of a finely divided solid form of a pharmaceuticalcomposition of the present invention and a dispersing agent. With eitherthe liquid or dry powder aerosol formulation, the formulation must beaerosolized. That is, it must be broken down into liquid or solidparticles in order to ensure that the aerosolized dose actually reachesthe mucous membranes of the nasal passages or the lung. The term“aerosol particle” is used herein to describe the liquid or solidparticle suitable for nasal or pulmonary administration, i.e., that willreach the mucous membranes. Other considerations, such as constructionof the delivery device, additional components in the formulation, andparticle characteristics are important. These aspects of nasal orpulmonary administration of a drug are well known in the art, andmanipulation of formulations, aerosolization means and construction of adelivery device require at most routine experimentation by one ofordinary skill in the art.

Often, the aerosolization of a liquid or a dry powder formulation forinhalation into the lung will require a propellant. The propellant maybe any propellant generally used in the art. Specific non-limitingexamples of such useful propellants are a chiorofluorocarbon, ahydrofluorocarbon, a hydrochlorofluorocarbon, or a hydrocarbon,including trifluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof.

Systems of aerosol delivery, such as the pressurized metered doseinhaler and the dry powder inhaler are disclosed in Newman, S. P.,Aerosols and the Lung, Clarke, S. W. and Davia, D. editors, pp. 197-22and can be used in connection with the present invention.

Liquid Aerosol Fornulations. The present invention provides aerosolformulations and dosage forms. In general such dosage forms contain apharmaceutical composition of the present invention in apharmaceutically acceptable diluent. Pharmaceutically acceptablediluents include but are not limited to sterile water, saline, bufferedsaline, dextrose solution, and the like.

The formulation may include a carrier. The carrier is a macromoleculewhich is soluble in the circulatory system and which is physiologicallyacceptable where physiological acceptance means that those of skill inthe art would accept injection of said carrier into a patient as part ofa therapeutic regime. The carrier preferably is relatively stable in thecirculatory system with an acceptable plasma half life for clearance.Such macromolecules include but are not limited to Soya lecithin, oleicacid and sorbitan trioleate, with sorbitan trioleate preferred.

The formulations of the present embodiment may also include other agentsuseful for pH maintenance, solution stabilization, or for the regulationof osmotic pressure. Aerosol Dry Powder Formulations. It is alsocontemplated that the present aerosol formulation can be prepared as adry powder formulation comprising a finely divided powder form ofpharmaceutical composition of the present invention and a dispersant.Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing pharmaceutical composition of thepresent invention (or derivative) and may also include a bulking agent,such as lactose, sorbitol, sucrose, or mannitol in amounts whichfacilitate dispersal of the powder from the device, e.g., 50 to 90% byweight of the formulation. The pharmaceutical composition of the presentinvention (or derivative) should most advantageously be prepared inparticulate form with an average particle size of less than 10 mm (ormicrons), most preferably 0.5 to 5 mm, for most effective delivery tothe distal lung.

In a further aspect, recombinant cells that have been transformed with anucleic acid encoding the immunosuppressant protein, an active fragmentthereof or a derivative thereof and that express high levels of thepolypeptide can be transplanted in a subject in need ofimmunosuppression. Preferably autologous cells transformed with ISP aretransplanted to avoid rejection; alternatively, technology is availableto shield non-autologous cells that produce soluble factors within apolymer matrix that prevents immune recognition and rejection.

Methods of Treatment, Methods of Preparing a Medicament. In yet anotheraspect of the present invention, methods of treatment and manufacture ofa medicament are provided. Conditions alleviated or modulated by theadministration of the present derivatives are those indicated above.

Dosages. For all of the above molecules, as further studies areconducted, information will emerge regarding appropriate dosage levelsfor treatment of various conditions in various patients, and theordinary skilled worker, considering the therapeutic context, age andgeneral health of the recipient, will be able to ascertain properdosing. In addition, where appropriate, the size of the tumor may berelevant.

A subject in whom administration of ISP or an active fragment thereof ora derivative thereof or an analog thereof, is an effective therapeuticregimen is preferably a human, but can be any animal. Thus, as can bereadily appreciated by one of ordinary skill in the art, the methods andpharmaceutical compositions of the present invention are particularlysuited to administration to any animal, particularly a mammal, andincluding, but by no means limited to, domestic animals, such as felineor canine subjects, farm animals, such as but not limited to bovine,equine, caprine, ovine, and porcine subjects, wild animals (whether inthe wild or in a zoological garden), research animals, such as mice,rats, rabbits, goats, sheep, pigs, dogs, cats, etc., avian species, suchas chickens, turkeys, songbirds, etc., i.e., for veterinary medical use.

In addition to rational design of agonists and antagonists based on thestructure of immunosuppressive protein of the present invention furthercontemplates an alternative method for identifying specific antagonistsor agonists and mimics using various screening assays known in the art.

Accordingly, any screening technique known in the art can be used toscreen for agonists, antagonists or mimics of ISP. The present inventioncontemplates screens for small molecules (i.e. compounds being less than3 Kd) or analogs and mimics, as well as screens for natural analogs thatbind to and agonize or antagonize HISP in vivo or mimic the role of HISPas an immune suppressor. For example, natural products libraries can bescreened using assays of the invention for molecules that agonize,antagonize, or mimic HISP activity.

Knowledge of the primary sequence of HISP can also provide clue as theinhibitors, antagonists, or mimics of the protein. Identification andscreening of antagonists for example is further facilitated bydetermining structural features of the protein, e.g., using X-raycrystallography, neutron diffraction, nuclear magnetic resonancespectrometry, and other techniques for structure determination. Thesetechniques provide for the rational design or identification of agonistsand antagonists.

Another approach uses recombinant bacteriophage to produce largelibraries. Using the “phage method” [Scott and Smith, 1990, Science249:386-390 (1990); Cwirla, et al., Proc. Natl. Acad. Sci., 87:6378-6382(1990); Devlin et al., Science, 249:404-406 (1990)], very largelibraries can be constructed (10 6-10.sup.8 chemical entities). A secondapproach uses primarily chemical methods, of which the Geysen method[Geysen et al., Molecular Immunology 23:709-715 (1986); Geysen et al. J.Immunologic Method 102:259-274 (1987)] and the method of Fodor et al.[Science 251:767-773 (1991)] are examples. Furka et al. [14thInternational Congress of Biochemistry, Volume 5, Abstract FR:013(1988); Furka, Int. J. Peptide Protein Res. 37:487-493 (1991)], Houghton[U.S. Pat. No. 4,631,211, issued December 1986] and Rutter et al. [U.S.Pat. No. 5,010,175, issued Apr. 23, 1991] describe methods to produce amixture of peptides that can be tested as agonists or antagonists.

In another aspect, synthetic libraries [Needels et al., Proc. Natl.Acad. Sci. USA 90:107004 (1993); Ohlmeyer et al., Proc. Natl. Acad. Sci.USA 90:10922-10926 (1993); Lam et al., International Patent PublicationNo. WO 92/00252; Kocis et al., International Patent Publication No. WO9428028, each of which is incorporated herein by reference in itsentirety], and the like can be used to screen for mimics for HISPaccording to the present invention.

Alternatively, assays for agents that promote immunosuppression can beperformed. The agents can be provided readily as recombinant orsynthetic polypeptides, for example.

The screening can be performed with cells that have been designed and/orselected for not expressing HISP. For example, the ability of such cellsto undergo apoptosis can be determined in the presence of agents whichare contained in a screening library, as described in the foregoingreferences. The agents can be selected for inducing such apoptosis.

In one example, a phage library can be employed. Phage libraries havebeen constructed which when infected into host E. coli produce randompeptide sequences of approximately 10 to 15 amino acids [Parmley andSmith, Gene, 73:305-318 (1988), Scott and Smith, Science, 249:386-249(1990)]. Specifically, the phage library can be mixed in low dilutionswith permissive E. coli in low melting point LB agar which is thenpoured on top of LB agar plates. After incubating the plates at37.degree. C. for a period of time, small clear plaques in a lawn of E.coli will form which represents active phage growth and lysis of the E.coli. A representative of these phages can be absorbed to nylon filtersby placing dry filters onto the agar plates. The filters can be markedfor orientation, removed, and placed in washing solutions to block anyremaining absorbent sites. The filters can then be placed in a solutioncontaining, for example, a radioactive fragment of HISP containing mostor all of its expressed coding region. After a specified incubationperiod, the filters can be thoroughly washed and developed forautoradiography. Plaques containing the phage that bind to theradioactive HISP fragment can then be identified. These phages can befurther cloned and then retested for their ability to hinder Tlymphocyte activation, for example. Once the phages have been purified,the binding sequence contained within the phage can be determined bystandard DNA sequencing techniques. Once the DNA sequence is known,synthetic peptides can be generated which represent these sequences.

The effective peptide(s) can be synthesized in large quantities for usein vivo models and eventually in humans to act as tumor suppressors. Itshould be emphasized that synthetic peptide production is relativelynon-labor intensive, easily manufactured, quality controlled and thus,large quantities of the desired product can be produced quite cheaply.Similar combinations of mass produced synthetic peptides have recentlybeen used with great success [Patarroyo, Vaccine, 10:175-178 (1990)].

Example 1 Production of Ntera2/D1

Ntera2/D1 cells (Layton Bioscience, Inc., Sunnyvale, Calif.) weremaintained in Dulbecco's minimal essential medium with nutrient mixtureF-12 (DMEM/F-12) with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/mlstreptomycin, and 10% (v/v) heat-inactivated fetal bovine serum (FBS)(Life Technologies, Gaithersburg, Md.), and incubated in a 37° C.,humidified, 5% CO₂ environment.

Example 2 Differentiation to hNT Neurons

Differentiation of Ntera2/D1 is described in detail by Andrews, 1984 andPleasure et al., 1992, which are hereby incorporated by reference.Briefly, Ntera2/D1 (2.times. 10⁶) were treated with 10 μM all-transretinoic acid (Sigma, St. Louis, Mo.) for 5-6 weeks. Mitoticnon-neuronal cells were inhibited with 1 μM cytosineβ-D-arabinofuranoside, 10 μM 5-fluoro-2′-deoxyuridine, and 10 μM1-β-D-ribofuranosyluracil (Sigma) for 1 week. Some retinoic acid-treatedNtera2/D1 cultures were exposed to 25 Gy γ-irradiation (¹³⁷Cs) toinhibit non-neuronal cells and were not treated with mitotic inhibitors,to ensure that trace amounts of mitotic inhibitors were not contributingto the immunosuppressive properties of hNT supernatant. DifferentiatedhNT neurons overlying the mixed cell culture were treated with 0.025%trypsin and 0.01% EDTA, dislodged, and replated at a density of 2−3×10⁶cells/ml in serum-free Opti-Mem medium (Gibco-BRL), or DMEM/F-12 10% FBSmedium, each supplemented with 100 U/ml penicillin, 100 μg/mlstreptomycin, and 2.0 mM L-glutamine, in flasks pretreated withpoly-D-lysine (Sigma) and coated with MATRIGEL® basement membrane matrix(Collaborative Research/Becton Dickinson, Bedford, Mass.). Cultures werefed serum-free Opti-Mem medium or DMEM/F-12 10% FBS medium for up to 6days, yielding hNT supernatant which was filter sterilized and stored at−20° C. for future analysis. These enriched hNT neuronal culturesconsisted of >90% neurons.

Some hNT neurons were cultured 4 days in the presence of 0.5 or 1.0 μMd, 1-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol(Matreya, Inc., Pleasant Gap, Pa.), a potent inhibitor ofglucosylceramide synthase and ganglioside shedding (Olshefski andLadisch, 1998; Felding-Habernann et al., 1990). An aliquot of hNTsupernatant was collected after the 4-day exposure. hNT neurons werewashed, suspended in fresh serum-free Opti-Mem medium, and aliquots ofhNT supernatant saved at 24, 48, and 72 hrs post-washing. Each aliquotwas analyzed for antiproliferative activity.

Some hNT neurons were cultured in the presence of 1.2 mg/mlN-g-monomethyl-L-arginine (Schweizerhall, Inc., Piscataway, N.J.), aninhibitor of nitric oxide synthase for 3 days and the hNT supernatantanalyzed for antiproliferative activity.

Supernatant from other cultures of human NCCIT embryonal carcinomacells, T98G glioblastoma cells, or THP-1 monocytic leukemia cells (ATCC)were also analyzed for anti-proliferative activity, but were found tohave very little (each eliciting less than a mean 1.5% suppression ofthe PHA assay (n=6), data not shown).

Example 3 MHC Genotype & Phenotype

Ntera2/D1 genomic DNA (75-125 ng/μl) was used as template for MHC classI and II analysis by sequence-specific primer polymerase chain reaction(Bunce et al., 1995) in MHC diagnostic plates (Pel-Freez ClinicalSystems, Deerbrook Trail, Wis.), and the amplified products analyzed byethidium bromide stained electrophoresis.

hNT MHC surface expression was detected using a complement-dependentcytotoxic technique in Terasaki tissue typing trays (One Lambda, Inc.,Canoga Park, Calif.). Briefly, 2×10⁶/ml hNT neurons were reacted withMHC antigen-specific monoclonal antibody (mAb) or known antisera, mixedwith rabbit complement, ethidium bromide and acridine orange, and thereaction stopped by hemoglobin-EDTA. Cell viability was scored, and theMHC phenotype determined. At least 2 mAb or 3 overlapping antisera wereused to define each MHC antigen.

The hNT neurons express MHC class I proteins. The amplified products ofpolymerase chain reactions using Ntera2/D1 genomic DNA as templateindicated a MHC class I and II genotype of A1 B8 Bw6 Cw7 DR3 DR52 (FIG.1). The surface expression of MHC molecules on hNT neurons was detectedusing a complement-dependent cytotoxic technique and limited to theclass I proteins A1 B8 Bw6. No surface expression of MHC class IIproteins was detected on hNT neurons.

Example 4 Isolation of PBMC

Peripheral blood mononuclear cells (PBMC) were isolated from the bloodof healthy human donors by layering over Accu-Prep (Accurate ChemicalCorp., Westbury, N.Y.). The interface band was collected, washed, thensuspended in either scrum-free Opti-Mem medium, or in RPM1-1640 with 10%FBS supplemented with 100 U/ml penicillin, 100 γg/ml streptomycin and2.0 mM L-glutamine (RPMI medium with 10% FBS).

Mixed Lymphocyte and Mixed Lymphocyte-Neuron Cultures. Mixed lymphocytecultures (MLC) consisted of 10⁵ responder and 10⁵ allogeneic stimulatorPBMC. Mixed lymphocyte-neuron cultures (MLNC) consisted of 10.sup.5responder PBMC and 10.sup.5 stimulator hNT neurons. MLC or MLNC wereestablished in triplicates, incubated for 4 days, pulsed with³H-thymidine (3H-TdR), harvested (Packard Instrument, Meriden, Conn.),and counted in a β-spectrometer (Packard Instrument). The uptake of³H-TdR by stimulator cells was prevented by prior 25 Gy γ-irradiation(¹³⁷Cs). The stimulation index of responder PBMC was determined bydividing the mean cpm of triplicate stimulated cultures by the mean cpmof triplicate control syngeneic cultures. Assays were prepared usingserum-free Opti-Mem medium, RPMI medium with 10% FBS, or a 1:2 finaldilution of hNT supernatant using either medium. Viability of PBMC inMLC and MLNC was assured by trypan blue dye exclusion on day 4.

hNT neurons do not stimulate PBMC proliferation. We first tested theimmunogenic potential of hNT neurons in vitro, by mixing hNT neuronswith allogeneic PBMC, and assessing for the DNA synthesis andproliferation of lymphocytes. In control MLCs involving 6 separatedonors, responder PBMC proliferated in the presence of unmatched,irradiated stimulator PBMC. with a mean stimulation index of 9.4±7.9(n=16). In spite of the surface expression of MHC class I proteins onhNT neurons, irradiated hNT neurons did not induce responder PBMC toproliferate in MLNC (FIG. 2A). hNT neurons were derived from Ntera2/Dcultures treated with RA, and exposed to either mitotic inhibitors or to25 Gy γ-irradiation (¹³⁷Cs) to eliminate non-neuronal cell growth. Ineither case, hNT neurons did not induce responder PBMC from 4 separatedonors to proliferate, with a mean stimulation index of only 0.2±0.1(n=12), significantly less than compared to control MLCs (p<0.01).Viability of PBMC in MLNC on day 4 was comparable to that of PBMC incontrol MLC, and routinely >90% in trypan blue dye exclusion assays.

hNT supernatant suppresses allogeneic MLC. To determine whether thisabsence of PBMC proliferation in the presence of hNT neurons wasattributable to a soluble factor expressed by hNT neurons, we addedsupernatant from hNT cultures to allogeneic MLC so that the resultantconcentration of hNT supernatant was 1:2. hNT supernatant from culturesmaintained with or without serum, and treated earlier with eithermitotic inhibitors or .gamma.-irradiation to eliminate non-neuronal cellgrowth, suppressed the proliferation of responder PBMC in allogeneicMLCs by more than 98% compared to control MLC (p<0.01), with a meanstimulation index of only 0.1±0.1 (n=9) (FIG. 2A). Viability of PBMC inMLC on day 4, cultured in either control medium or in the presence of1:2 hNT supernatant, was comparable, and >90% as indicated by trypanblue dye exclusion.

Example 5 T-Cell Proliferation and IL-2 Production

The accessory cell-dependent mitogens PHA at 1:50 or 1:250, orconcanavalin A at 1:20, which cross-link the T-cell receptor, were usedto activate triplicate cultures of 105 PBMC. Assays were prepared usingserum-free Opti-Mem medium, RPMI medium with 10% FBS, or 1:2 hNTsupernatant, incubated for 48 hours, pulsed, harvested, and the uptakeof ³H-TdR determined. Viability of PHA-stimulated PBMC was assured bytrypan blue dye exclusion on day 2.

Recombinant human IL-2 (rhIL-2) (R&D Systems, Minneapolis, Minn.) at5-500 ng/ml was added to some PHA-stimulated PBMC cultures either 1 hrprior to, upon, or 24 hrs after addition of PHA.

Phorbol 12-myristate 13-acetate (PMA) can bind directly to and activateprotein kinase C, leading to DNA synthesis and T-lymphocyteproliferation. Calcium ionophores such as ionomycin can increase thecytosolic calcium concentration of T-cells and lead to T-cell division.PMA and ionomycin act synergistically to stimulate IL-2 production andthe proliferation of T-cells independent of an accessory cell influence.PMA at 10 ng/ml and ionomycin at 100 ng/ml were used to activatetriplicate cultures of 10.sup.5 PBMC. Assays were prepared using eitherserum-free Opti-Mem medium, or 1:2 hNT supernatant, incubated, pulsed,harvested, and the uptake of ³H-TdR determined.

The influence of hNT supernatant on the production of IL-2 wasdetermined by comparing IL-2 levels in the supernatant of PHA-stimulatedPBMC cultures containing either Opti-Mem medium or 1:2 hNT supernatant.Expressed levels of IL-2 prior to, and 4, 15, 24, and 48 hours after PHAstimulation were determined by ELISA (R&D Systems).

The hNT supernatant suppresses T-cell proliferation and IL-2 production.hNT supernatant with or without serum, from cultures treated earlierwith either mitotic inhibitors or γ-irradiation to eliminatenon-neuronal cell growth, significantly suppressed PHA-stimulatedproliferation 87±12% (n=20), and concanavalin A-stimulated proliferation79±19% (n=8), each (p<0.01) (FIG. 2B). Dilutions of hNT supernatantsuppressed PHA-stimulated proliferation in a dose-dependent manner, whentested at 1:2, 1:20, and 1:200 final concentrations, causing a mean 93%,62%, and 21% suppression, respectively. That hNT supernatant could alsoblock ongoing T-cell proliferation was demonstrated by adding hNTsupernatant up to 48 hrs after PHA stimulation, with a mean suppressionof 84.+−0.1% (n=6). This suppression of mitogen-driven proliferation ofPBMC by hNT supernatant was mediated without a reduction in PBMCviability, determined 2 days after PHA stimulation, with >90% cellsviable by trypan blue exclusion.

Control PHA-stimulated cultures of PBMC expressed 621±41 pg/ml IL-2, 48hours after mitogen stimulation. In contrast, PHA-stimulated PBMCcultured in the presence of hNT supernatant expressed less IL-2, withmean IL-2 levels detected by ELISA at 48 hours post-stimulation of only223±111 pg/ml (p<0.01) (FIG. 3).

Adding supplemental IL-2 to the PHA-stimulated assay did not activatethe “quiescent” PBMC cultured in the presence of hNT supernatant. Asexpected, when 5-50 ng/ml rhIL-2 was added to control assays 1 hr beforePHA stimulation, T-cell proliferation was increased a mean 47±12% (n=2)above levels induced by PHA alone (FIG. 2B). In contrast, addingsupplemental rhIL-2 did not reverse the T-cell suppressive activity ofthe hNT supernatant, which persisted in suppressing the proliferation ofPHA-stimulated PBMC a mean 97±3% (n=3) (p<0.01) (FIG. 2B).

PHA activation of T-cell proliferation results in cross-linkage of theT-cell receptor-CD3 (TCR-CD3) complex and is influenced by accessorycell signals. To determine whether hNT supernatant could suppress thedirect activation of T-cells by PMA or ionomycin, independent of anaccessory cell influence and independent of TCR-CD3 interactions, hNTsupernatants were added to PBMC cultures stimulated with 10 ng/ml PMA,or 100 ng/ml ionomycin, or both. hNT supernatant consistently andsignificantly suppressed the direct activation of T-cell proliferationby PMA, ionomycin, or both by 99±1% (n=3), each (p<0.001).

Example 6 Cell Cycle Analysis

To further demonstrate that hNT supernatant did not lower PBMC viabilityin immunoassays, propidium iodide stained PBMC were evaluated by flowcytometry, 48 and 72 hrs after PHA-stimulation. PBMC were stimulatedwith PHA in Opti-Mem medium or 1:2 NT2N-CM, harvested at 48 or 72 hrs,washed, stained with the nucleic acid binding dye propidium iodide(Sigma), and analyzed for DNA content by flow cytometry (fluorescenceintensity at 600-650 nm). The proportion of cells in each distinct phaseof the cell cycle was calculated with ModFit LT 2.0 software (VeritySoftware House, Topsham, Minn.).

Cell cycle analysis revealed that hNT supernatant did not reduce PBMCviability compared to controls (90.8±1.7%), that hNT supernatant heldPHA-stimulated PBMC in a growth arrested, G₀/G₁ phase (97±2%), and thatthe proportion of PBMC in either the S phase or G₂/M phase was reducedby as much as 92% to a mean of only 1.5±0.7% (n=2). The proportions ofPBMC undergoing apoptosis, necrosis, and the amount of cellular debrisin the modeled events were not different regardless of treatment.

Example 7 Detection and Immunoprecipitation of TGF-β

The hNT supernatant was tested for the presence of TGF-β (R&D Systems)and interleukin-10 by ELISA (Genzyme, Cambridge, Mass.), and forprostaglandin-E2α, vasoactive intestinal peptide (Peninsula Labs, Inc.,San Carlos, Calif.), and α-melanocyte stimulating hormone (PhoenixPharmaceuticals, Belmont. Calif.) by EIA.

To remove TGF-β from hNT supernatant, neutralizing anti-TGF-β mAb 240 at0.5-10.0 μg/ml (R&D Systems) was added and reacted overnight at 4° C.under rotating conditions. An excess of protein G Sepharose (AmershamPharmacia Biotech, Piscataway, N.J.) was added, and reacted for 10 hoursat 4° C. The mixture was centrifuged at 2000×g for 10 minutes, and thesupernatant tested for anti-proliferative activity. Immunoprecipitationof TGF-β was verified by ELISA. In other experiments, 0.01-10.0 μg/mlneutralizing anti-TGF-β mAb 240 was added directly to PHA-stimulatedPBMC assays.

We confirmed that purified TGF-β at concentrations detected in somesamples of hNT supernatant (100-1000 pg/ml), could suppressproliferation of the IL-2- and IL-4-dependent helper T-cell line HT-2(Ho et al., 1987) by a mean 40±3.5%, but this TGF-β-mediated suppressionwas less than the 90±9% suppression mediated by the hNT supernatant(n=3) (p<0.01) (data not shown). We also confirmed that 0.1-0.4 μg/mlmAb 240 could neutralize 53% of the antiproliferative effect of 250μg/ml purified TGF-β on HT-2 cells, reducing it from 40±3.5% to 19±3%suppression (n=3) (data not shown). In contrast, as described below,immunoprecipitation of TGF-β did not reduce the suppressive activity ofthe hNT supernatant.

Although TGF-β was detected in 81% of hNT supernatant aliquots at a meanlevel of 753±512 pg/ml, aliquots of hNT supernatant without detectableTGF-β (threshold >0.1 ng/ml) fully suppressed the PHA assay 94.+−0.5%(n=3), data not shown. TGF-β was removed from samples of hNT supernatantusing a neutralizing anti-TGF-β mAb and an excess of protein GSepharose. Complete immunoprecipitation of TGF-β, verified by ELISA, didnot reduce the immunosuppressive activity of the hNT supernatant, with97±3% suppression of the PHA assay prior to immunoprecipitation, and93±1% suppression after TGF-β immunoprecipitation (n=5), data not shown.In other experiments, addition of 0.01-10.0 μg/ml neutralizinganti-TGF-β mAb 240 directly to the PHA assay did not alter thesuppressive activity of the hNT supernatant, with 97±1% suppression ofthe PHA assay retained with mAb treatment (n=7).

Example 8 Characterization of the hNT Immunosuppressive Protein

hNT supernatant was concentrated using ultrafiltration (YM10, Amicon,Danvers, Mass.) and fractionated using Sephacryl S-300 HR gel in a2.5×95 cm column (Amersham Pharmacia Biotech). Each 4 ml fraction wasassessed for protein content at 280 nm. Groups of five fractions werepooled, diluted 1:20, and tested for ability to suppress PHA-inducedPBMC proliferation.

hNT supernatant, or the immunosuppressive gel filtration fractions, weretreated with either heat (56° C. for 30 minutes), or to pH 2 or pH 11,or mixed with trypsin- or carboxypeptidase A-coated agarose beads(Sigma) each for 60 minutes, or incubated with Vibrio choleraeneuraminidase-coated agarose beads (Sigma) for 2 hours to eliminategangliosides, then evaluated and tested for retained suppressiveactivity in a PHA-stimulated assay.

To determine whether the antiproliferative factor(s) could bind toaffinity resins, either 250 mg/mi Heparin-Sepharose CL-B gel, or BlueSepharose gel (Amersham Pharmacia Biotech) were separately combined witheither hNT supernatant or the active gel filtration fractions at ratiosof 1:1 to 4:1. Each separate mixture was centrifuged, and thesupernatants and washes containing unbound proteins were retained foranalysis. The bound protein was eluted from each gel with 0.25 M-1.5 MNaCl. Eluted fractions were desalted with PD-10 Desalting Columns(Amersham Pharmacia Biotech). The unbound and eluted fractions weretested for retained suppressive activity in PHA-stimulated assays.

Active gel filtration fractions were also tested for binding toSepharose Fast Flow resins comprised of weak (DEAE) and strong (Q) anionexchangers, and weak (CM) and strong (SP) cation exchangers (PharmaciaBiotech). Bound fractions were removed from exchange resins using0.25-1.5 M NaCl in 50 mM HEPES buffer. The eluted fractions weredialyzed against 0.15 M NaCl in 0.5 M HEPES. The unbound fraction andeluted fractions were each tested for suppression of PHA-induced T-cellproliferation.

The peak active gel filtration fraction was used for isoelectricfocusing using the Bio-Rad Rotofor system, and a broad (pH 3-10)ampholyte range, followed by a narrow (pH 4-6) ampholyte range. Afterfocusing with 1.5 ml ampholyte (pH 4-6, 40% w/v) and 45.5 ml doublydistilled water for 60 minutes at 15 watts and 4° C., the activeisoelectric fraction was identified. This active isoelectric fraction at2.1 mg in a 3.0 ml volume was then focused for an additional 3 hoursusing the same conditions. Ampholytes were removed by the addition of0.25 ml of 5 M NaCl per ml, and fractions were exhaustively dialyzedagainst 50 mM HEPES, pH 7.2, containing 150 mM NaCl for 22 hours at 4°C. Immunosuppressive activity was detected using the PHA-stimulatedassay.

hNT neurons were cultured for 4 days in the presence of 0.5 or 1.0 μM d,1-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol, a potentinhibitor of glucosylceramide synthase and ganglioside shedding. Analiquot of hNT supernatant was collected after the 4-day exposure,neurons were washed, suspended in fresh medium, and aliquots of hNTsupernatant saved at 24, 48, and 72 hrs post-washing. In spite of thisganglioside-inhibiting treatment of neuron cultures, the suppressiveactivity of the hNT supernatant was retained, with 93±10% inhibition ofthe PHA assay (n=4), data not shown.

hNT neurons were cultured in the presence of 1.2 mg/mlN-g-monomethyl-L-arginine, an inhibitor of nitric oxide synthase, for 3days and found that the suppressive activity of the hNT supernatant wasfully retained (data not shown).

No evidence was found indicating that the hNT supernatant had T-cellsuppressive levels of prostaglandin-E2α (assay threshold of >39 pg/ml),α-melanocyte stimulating hormone (>1.3 ng/ml), vasoactive intestinalpeptide (>0.7 ng/ml), or IL-10 (>8.5 pg/ml).

These findings encouraged efforts to concentrate, characterize, andpurify the suppressive activity of hNT supernatant. The PHA-activatedT-cell proliferation assay was used as a simple and reliable indicatorof T-cell reactivity. As shown in Table 1, hNT supernatant lost most ofits suppressive activity when heated or exposed to low pH 2, or treatedwith carboxypeptidase A. hNT supernatant exposed to high pH 11, ortreated with trypsin retained approximately 48% residual T-cellsuppressive activity. The antiproliferative activity of hNT supernatantdid not bind to Heparin-Sepharose CL-B gel, with unbound fractionssuppressing T-cell proliferation a mean 99±0% (n=5).

hNT supernatant was concentrated using YM10 ultrafiltration, and theconcentrate fractionated using a Sephacryl S-300 HR gel. Each fractionwas assessed for protein content, and pools of five fractions werediluted 1:20 and tested for suppressive activity. Pooled fractions werefound to suppress T-cell proliferation over a molecular mass range ofapproximately 40-100 kDa (FIG. 4). The peak immunosuppressive activefraction had approximately 7.7 μg/ml of total protein.

TABLE 1 Immunosuppressive Properties of hNT Supernatant Suppression ofT-cell Proliferation* Treatment of hNT Supernatant None ++++ 56° C. + pH2 + pH 11 ++ Trypsin ++ Carboxypeptidase A + Heparin Sepharose ++++ GelFiltration Fraction ~40-100 KDa ++++ Treatment of Fraction ~40-100 kDaBlue Sepharose ++++ Trypsin + Carboxypeptidase + Neuraminadase ++++Cation Exchange Resin ++++ Anion Exchange Resin + *% Suppression ofPHA-stimulated PBMC proliferation: ++++, 80-100%; +++, 60-79%; ++,40-59%; +, 10-39%; −, none.

The antiproliferative activity of the peak active gel filtrationfractions was not degraded by exposure to Vibrio choleraeneuraminidase-coated agarose beads to eliminate gangliosides (Table 1).In contrast, protease treatments using trypsin or carboxypeptidaseeliminated most of the suppressive activity of these fractions (Table1). Although 79% of the total protein in the peak active fraction boundto the albumin-binding resin Blue Sepharose, the unbound fractioncontinued to suppress the proliferation of T-cells a mean 75±5% (n=2),suggesting that the immunosuppressive protein expressed by hNT neuronswas not carried by albumin.

The peak active fraction was tested for binding to weak or strong anionor cation exchangers. Bound fractions were eluted from exchange resinsand dialyzed against HEPES buffer, then bound and unbound fractions weretested for suppressive activity. The T-cell suppressive activity of thepeak active fraction consistently bound to anion, but not to cationexchanger resins, indicating a net anionic charge. Unbound fractions ofweak (CM) and strong (SP) cation exchange resins continued to suppressT-cell proliferation a mean 77±31% and 99±0%, respectively (n=2) (Table1). In contrast, unbound fractions of weak (DEAE) anion exchange resinsuppressed T-cells only 10±17%, and unbound fractions of strong (Q)anion exchange resin had no residual T-cell suppressive activity (n=3).Bound proteins that were eluted from the anion exchangers suppressed theproliferation of T-cells 99% (data not shown).

We next used the peak active gel filtration fraction for isoelectricfocusing. Preliminary broad range isoelectric focusing indicated thatthe immunosuppresive protein had an isoelectric point of approximately5. Consequently, we focused the peak active fraction using a narrowampholyte pH range of 4-6. Of the twenty isoelectric fractionscollected, only fraction #10 suppressed either the PHA orPMA/ionomycin-induced proliferation of T-cells more than 70%, each(p<0.01) indicating that the hNT immunosuppressive protein had anisoelectric point of 4.8 (FIG. 5).

Current research employs a purification scheme that utilizes preparativepolyacrylamide gel electrophoresis of the active isoelectric fraction#10. It incorporates Blue Sepharose removal of albumin whose isoelectricpoint of 4.9 causes it to elute in close proximity to the activeprotein, and the concentrating effect of Q Sepharose columns using 50 mMethanolamine pH 9 buffer. We are concurrently testing whether stabilityof the protein is improved by incorporating the use of β-mercaptoethanoland various concentrations of glycerol in the eluting buffer.

Although others have demonstrated using broad, nonspecific antisera thatdifferentiation of Ntera2/D1 cells to hNT neurons results in theexpression of some unspecified MHC class I and β-2 microglobulinmolecules (Segars et al., 1993), we determined the specific MHC genotypeand phenotype of these cells. In spite of the demonstrated surfaceexpression of MHC class I proteins A1 B8 Bw6, hNT neurons did notactivate the proliferation of allogeneic immune cells in vitro. Thislack of allogeneic T-lymphocyte activation by hNT neurons was notattributable to low constitutive MHC class I expression on the surfaceof the hNT neurons, since hNT supernatants potently suppressed T-cellproliferation in a dose-dependent manner.

A unique hNT neuron immunosuppressive protein with a molecular mass of40-100 kDa, an isoelectric point 4.8, and a net anionic charge wasidentified. The abrogating effect of this hNT protein on T-cellactivation and proliferation was direct, and not mediated through theT-cell receptor-CD3 complex, or via altered accessory cell signals. Itcaused a significant reduction in the level of IL-2 expressed byT-cells, and supplemental IL-2 could not override its immunosuppressiveeffect. The quiescent T-cells were viable and arrested in the G0/G₁phase of the cell cycle.

Our initial evaluations of the immunosuppressive properties of the hNTsupernatant were guided by precedents, which showed that retinoic acidtreatment of other embryonal carcinoma cell lines can increase sheddingof gangliosides (Chen et al., 1989; Osanai et al., 1997), or expressionof TGF-β (Rizzino et al., 1983), which can be immunosuppressive invitro.

We developed multiple lines of evidence that dismissed the potentialthat the hNT immunosuppressive effect could have been attributed togangliosides shed from the hNT neurons. Inhibition of T-cellproliferation and IL-2 production by ganglioside-enriched supernatantsfrom brain lipid homogenates is found in the lipid-enriched andprotein-depleted fraction (Irani et al., 1996; Irani et al., 1997). Incontrast, hNT supernatant was used directly in immunoassays, without aselective lipid extraction enriching for gangliosides, and effectivelysuppressed T-cell proliferation. Pretreating brain ganglioside-enrichedsupernatant with neuraminidase eliminates the inhibitory effect of thebrain-derived supernatant on T-cells (Irani et al, 1997). In contrast,neuraminidase pretreatment of the hNT supernatant peak active gelfiltration fraction did not reduce its suppressive effect on T-cellproliferation. Brain tumor cells can shed gangliosides in vitro and invivo, potentially in monomeric form (<2 kDa), bound to albumin (68 kDa),or as micelles (130 kDa) (Kong et al., 1998; Valentino et al., 1990). Incontrast, the T-cell suppressive protein in hNT supernatant ranged inmass between 40-100 kDa, and did not segregate with albumin or otherBlue Sepharose-bound proteins. Tumor cell shedding of gangliosides invitro can be inhibited 83% by culturing cells in the presence of 1.0 μMd, 1-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol, aninhibitor of glucosylceramide synthase (Felding-Habermann et al., 1990).In contrast, culturing hNT neurons in the presence of 0.5 or 1.0 μM d,1-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol did notreduce the suppressive effect of hNT supernatant on T-cellproliferation.

Similarly, several lines of evidence dismiss the potential thatTGF-.beta. was responsible for the dramatic suppression of T-cellproliferation by hNT supernatant. First, hNT supernatant withoutdetectable levels of TGF-β (threshold >0.1 ng/ml) suppressed T-cellproliferation. Immunoprecipitation of TGF-β from hNT supernatant thathad detectable TGF-β levels using neutralizing anti-TGF-β mAb did notsubstantially reduce the T-cell suppressive activity of the hNTsupernatant. Prior reports have shown that although a 1.0 ng/ml dose ofTGF-β could suppress 50% of either a PHA or PMA/ionomycin-stimulatedT-cell proliferation when added up to 6 hours after stimulation, thissuppressive effect of TGF-β was lost 16 hours after T-cell activation(Ahuja et al., 1993). In contrast, hNT supernatant suppressed 84% of aPHA-driven T-cell proliferation, even up to 48 hours after T-cellstimulation. Further, TGF-β is a heparin-binding protein (McCaffrey etal., 1992), but the hNT immunosuppressive protein did not segregate withheparin-bound proteins. Finally, the peak active gel filtration fractionof hNT supernatant that profoundly suppressed T-cell proliferation hadno detectable TGF-β by ELISA.

We sought but could find no evidence of other potential co-mediators ofthe T-cell suppressive effect of the hNT immunosuppressive protein.Although T-cell proliferation in vitro may be modulated by neuropeptides(e.g., vasoactive intestinal peptide) (Sun and Ganea, 1993; Nio et al.,1993), or annexin II (Nygaard et al., 1998), or neurotransmitters (e.g.,dopamine) or their metabolites (e.g., homovanillic acid), our evidencesuggested that the observed T-cell suppressive effect of hNT neurons andsupernatant was attributable to the expression of a single anionicprotein with an isoelectric point of 4.8.

Studies of Ntera2/D1 cells and their hNT neuron derivative maycontribute to the development of a transfectable and transplantableneuron with both therapeutic and protective features. Assuming that thehNT immunosuppressive protein can modulate immune responsiveness invivo, then hNT neuron grafts may be both therapeutic andself-protective, either alone or as co-grafts with other cells. hNTneurons may serve as a model of the neuronal regulation of immuneprivilege within the CNS.

This novel T-lymphocyte suppressive hNT protein has broad applicationsin preventing graft rejection in transplantation settings, in thetreatment of autoimmune diseases, and in the suppression of severeallergic responses. Further, its neuronal origin introduces thelikelihood that it may represent a novel class of immunomodulators,which are responsible for the maintenance of CNS immune privilege.

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What is claimed is:
 1. An isolated human immunosuppressant protein(HISP) using the steps comprising: a) growing a culture of hNT neuronalcells; b) obtaining supernatant from the hNT cell culture; c)concentrating the supernatant; d) exposing the supernatant to gelelectrophoresis to produce isoelectric fractions; e) collecting a peakimmunosuppressive active isoelectric fraction; and f) collecting theprotein extract from the fraction wherein the protein extract has anisoelectric point of about 4.8, a molecular weight of 40-100 kDa andexhibits at least one immunosuppressive activity selected from the groupconsisting of suppressed T-cell activation, suppressed T-cellproliferation, and suppressed production of IL-2 by T-cells.
 2. Theprotein extract of claim 1 isolated using the steps, further comprising:purifying the peak immunosuppressive active isoelectric fraction byremoving albumin on an albumin-binding column; and concentrating theactive isoelectric fraction on an anionic column; column prior tocollecting the protein extract.
 3. The protein extract of claim 1,wherein the supernatant is concentrated by ultrafiltration.
 4. Theprotein extract of claim 1, wherein the active isoelectric fractionsuppresses T-cell proliferation and suppresses production of IL-2 byT-cells by at least about 70%.
 5. An isolated human immunosuppressantprotein (HISP) extract using the steps comprising: a) growing a cultureof hNT neuronal cells; b) obtaining supernatant from the hNT neuronalcell culture; c) concentrating the supernatant; d) exposing thesupernatant to gel electrophoresis to produce isoelectric fractions; e)purifying an isoelectric fraction exhibiting peak immunosuppressiveactivity; and f) collecting the protein extract having a molecularweight of 40-100 kDa and an isoelectric point of about 4.8 from thefraction wherein the protein extract exhibits at least oneimmunosuppressive activity selected from the group consisting ofsuppressed T-cell activation, suppressed T-cell proliferation, andsuppressed production of IL-2 by T-cells.
 6. The protein extract ofclaim 5, wherein the supernatant is concentrated by ultrafiltration. 7.The protein extract of claim 5, wherein the active isoelectric fractionsuppresses T-cell proliferation and suppresses production of IL-2 byT-cells by at least about 70%.
 8. The protein extract of claim 5 whereinthe isoelectric fraction is purified by removing albumin through the useof an albumin-binding column.
 9. The protein extract of claim 5 isolatedusing the steps, further comprising: concentrating the activeisoelectric fraction on an anionic column prior to collecting theprotein extract.
 10. An isolated human immunosuppressant protein (HISP)extract using the steps comprising: a) obtaining supernatant from hNTneuronal cell culture; b) concentrating the supernatant usingultrafiltration; c) exposing the supernatant to preparativepolyacrylamide gel electrophoresis to produce isoelectric fractions; d)collecting a peak immunosuppressively active isoelectric fractionwherein immunosuppression is determined by PHA-stimulated assay; e)purifying the active isoelectric fraction by removing albumin on analbumin-binding column; f) concentrating the active isoelectric fractionon an anionic column; and g) collecting the isolated protein extracthaving an isoelectric point of about 4.8, a molecular weight of about40-100 kDa and exhibits at least one immunosuppressive activity selectedfrom the group consisting of suppressed T-cell activation, suppressedT-cell proliferation, and suppressed production of IL-2 by T-cells. 11.The protein extract of claim 10, wherein the active isoelectric fractionsuppresses T-cell proliferation and suppresses production of IL-2 byT-cells by at least about 70%.
 12. The protein extract of claim 10,further comprising treating the isoelectric fractions obtained in step dby a method selected from the group consisting of exposing the fractionsto pH 11 environment, and incubating the fraction with Vibrio choleraeneuraminidase-coated agarose beads for 2 hours.